Bone marrow has been considered as a source of musculoskeletogenic (“MSG”) components for producing autologous graft materials useful in the repair/regeneration of musculoskeletal tissues such as bone, cartilage and tendon. Bone marrow aspirate (“BMA”) is typically obtained from the patient during surgery by well known techniques and includes the following components set out in Table I below:
TABLE IBMA ComponentVolume FractionPlasma40-45 vol %Buffy Coat Fraction (BCF) 1-10 vol %Red Blood Cells 45-50 vol %.
The BCF comprises all of the nucleated bone marrow cells (“NBMC”), platelets, proteins and molecules contained within the density band of materials residing between the serum and red blood cell portions of the BMA, as determined by conventional centrifugation of whole BMA. The NBMC component of the BCF typically comprises the following compliment of cell types and approximate concentrations as set out in Table II:
TABLE IIRelativeNativeAbsoluteConcentrationNative(Approximate)Concentration.(cells/total(Approximate)NBMC TypeNBMC cells)(cells/ml BMA)Musculoskeletal<1%<200,000 Precursor CellsMSPCsNucleated95-99%20 × 106HematopoiticCells (HCs)Reticulocytes<0.1% <20,000 (RCs),Endothelial<0.1% <20,000.Cells (ECs)
In a first conventional method of using bone marrow for its osteogenic capacity, whole or “fresh” bone marrow is either used directly as a graft material or is combined with a matrix material to produce a bone graft composite. For example, Harada, Bone 9 (1988) 177-183, disclosed a composite comprising whole BMA within a porous matrix of demineralized bone matrix (DBM) contained within a diffusion chamber. However, the diffusion chamber has a semi-permeable membrane that allows the passage of nutrients, and so prevents the influx of cellular components and vasculature critical to osteogenesis. Moreover, as the success of this procedure depends in part upon the native levels of MSPCs in the bone marrow, and such native levels of MSPCs in the patient's bone marrow can sometimes be depleted, the widespread utility of this procedure is limited. Moreover, even at relatively normal native levels of MSPCs, these cells are relatively scarce in fresh bone marrow and so the osteogenic potential of whole bone marrow per se is thereby limited.
In a second conventional method, plasma is removed from whole bone marrow, and the remaining mixture comprising the BCF and red blood cells is either used directly as a graft material or combined with a matrix material to produce a bone graft composite. For example, Ohgushi, J.Biomed. Mat.Res. (1990), 24:1563-70 disclosed centrifuging BMA, and using the remaining red cell/BCF fraction as an interstitial fluid within a porous matrix of HA or TCP. As plasma comprises about 45 volume percent (“vol %”) of bone marrow aspirate, this method produces only slightly elevated levels of MSPCs (i.e., less than a 2-fold increase) relative to the native level of MSPCs in the fresh bone marrow. In addition, the suspension essentially lacks the soluble or insoluble factors found in plasma such as albumin. Lastly, the presence of red blood cells (“RBCs”) in this composition may also cause inhibition of MSPC activity through steric hinderance of surface accessibility and high local iron concentrations following RBC lysis.
In a third conventional method, the buffy coat of the BMA is isolated from the plasma and red blood cell fractions. For example, Connolly et al., JBJS (1989) pp. 684-691, sought to “optimize” the osteogenic potential of BMA, and disclosed isolating fractions of BMA and then using those fractions as graft material in diffusion chambers. Connolly used the following isolation methods:                a) simple centrifugation followed by removal of the supernatant (i.e., serum) fraction,        b) isopyknic centrifugation, followed by separate removal of the light cell (buffy coat) and red cell fractions, and        c) unit gravity centrifugation, followed by separate removal of the light cell (buffy coat) and red cell fractions.Although Connolly reported that the concentrated light cell (buffy coat) fraction produced by isopyknic centrifugation yielded the greatest level of calcium production within the diffusion chamber, Connolly chose the combined red cell/light cell fraction produced by simple centrifugation (i.e., light cell and red cell fractions) for further study. Moreover, Connolly did not provide a porous substrate carrier material within the diffusion chamber. Lastly, Connolly's examples that utilized the BCF also eliminated the factors present in the plasma fraction of the BMA.        
In a fourth conventional method the isolated buffy coat is further fractionated. For example, Budenz et al., Am.J.Anat., 159 (1980), pp. 455-474, discloses isolating fractions of the BCF of bone marrow aspirate in high concentrations, and inserting that concentrated fraction into a diffusion container which is then implanted into rats. The limitations associated with diffusion chambers has been discussed above. Budenz does not disclose using the entire BCF fraction in toto. Lastly, Budenz does not disclose a porous substrate carrier material within the diffusion chamber.
In a fifth conventional method, an enriched fraction of MSPCs (relative to all other NMBCs) is combined with a matrix material to produce a bone graft. MSPCs can be enriched by a variety of well-known methods. For example, U.S. Pat. No. 6,049,026 (“Muschler '026”) discloses passing bone marrow aspirate through a matrix capable of selectively retaining MSPCs. This process produces a composite having enriched amounts of MSPCs (i.e., up to 2.8-fold higher than the native MSPC level found in the same volume of autologous bone marrow). However, this composite is also devoid of the cells, molecules and proteins present in BMA that are not retained by the substrate, and is depleted of other constituents found in BMA, which do not have a high affinity for the substrate. In addition, the process disclosed in Muschler '026 for enriching the MSPCs is inefficient, routinely failing to capture between about 33% and 56% of the MSPCs present in the BMA. Moreover, Muschler discloses optionally washing the MSPC-laden substrate in order to remove any cells which have been only loosely retained, thereby reducing even further the presence of cells which do not have a high affinity for the substrate. Muschler discloses optionally adding to the composite various discrete bioactive constituents such as platelets, cell adhesion molecules (such as collagens), growth factors (such as BMPs), antibodies (such as STRO-1).
Some investigators disclosed in vitro culturing of whole or fractionated BMA in an effort to obtain a plentiful and pure population of MSPCs. For example, Majors. J. Orthop. Res. (1997) 15:546-557, disclosed isolating the BCF of the BMA by centrifugation, culturing the BCF to produce an enriched MSPC population, and staining the MSPCs as a means for assaying the osteoblastic progenitor population within BMA.
PCT Published Patent Application No. 97/40137 (“Kadiyala”) discloses compositions and methods for augmenting bone formation by administering isolated human mesenchymal stem cells with a ceramic material or matrix or by administering human mesenchymal stem cells; fresh, whole marrow or combinations thereof in a resorbable biopolymer that supports their differentiation into their osteogenic lineage. Kadiyala contemplates the delivery of (i) isolated, culture expanded, human mesenchymal stem cells; (ii) freshly aspirated bone marrow; or (iii) their combination in a carrier material or matrix to provide for improved bone fusion area and fusion mass, when compared to the matrix alone. In Example V, discloses a composition comprising a collagen/ceramic composite mixed 50:50 with fresh bone marrow nucleated cells that had been concentrated ten-fold by centrifugation and buffy coat isolation (BMC). The procedure required for producing the culture-expanded, purified MSPCs is a long and arduous one (often requiring about about 21 to 56 days), and so can not be performed intra-operatively. U.S. Pat. No. 5,914,121 (“Robey”) discloses a composition comprising cultured MSPCs and HA/TCP powder, and optionally adding commercially-prepared fibrinogen and thrombin to the composition for the purpose of making fibrin glue.
A few investigators have reported supplementing porous matrices containing concentrated MSG fractions with whole BMA. For example, Walsh, “Autologous Growth Factor Gel (AGF) And Spinal Fusion” 47th Annual Meeting, ORS, February 2001, discloses a graft material comprising a HAP porous matrix, PRP and whole BMA. However, Walsh does not disclose a concentrated, physiologic fraction of fractionated bone marrow aspirate BMA, only whole BMA.
Matsukura, “Concentration of Bone Marrow Derived Osteoprogenitors for Spinal Fusion”, Am. Soc. Bone. Min. Res. 22nd Annual Meeting Abstracts, September 2000, discloses a graft material comprising an enriched fraction of MSPCs, whole bone marrow and a porous matrix. Matsukura does not disclose a concentrated, physiologic fraction of fractionated bone marrow aspirate BMA. The enriched fraction of MSPCs taught in Matsukura is not a suspension and so is depleted of the soluble constituents present in the corresponding physiologic fraction of BMA having high levels of MSPCs.
A US Patent Application entitled, “Composite Bone Marrow Graft Material With Method and Kit” (“Muschler II”) discloses a composite bone marrow graft material comprising a porous biocompatible implantable matrix, an enriched population of progenitor cells (MSPCs) and a clot material. The clot material can be a blood clot formed from blood, a bone marrow clot, a platelet gel, a platelet concentrate, fibrin clot or a fibrin glue. Since the enriched population of MSPCs is formed by the method taught in Muschler I and so (like Matsukura) is depleted of the soluble constituents present in the corresponding physiologic fraction of BMA having high levels of MSPCs, Muschler II does not disclose a concentrated, physiologic fraction of fractionated bone marrow aspirate BMA.
In sum, the conventional technologies either:                a) use whole marrow as a source of MSPCs, and so suffer from low MSPC concentrations (such as Walsh),        b) seek to enrich MSPCs by wholly eliminating other MSG constituents found in the BMA, and so do not have some of the supplemental MSG constituents present in BMA (such as Muschler I),        c) introduce isolated supplemental MSG constituents into composites having enriched levels of MSPCs, and so have only partially provided the supplemental MSG constituents present in BMA (such as Muschler I), or        d) add merely whole BMA into composites having enriched levels of MSPCs and so have only unenhanced levels of the supplemental MSG constituents (such as Muschler II and Matsukura)        
Moreover there is only a sporadic appreciation in the prior art of the advantages of combining MSG fractions with a porous matrix. For example, there is no disclosure in the prior art of a combination of a physiologic fraction of BMA in combination with a matrix and supplemented with whole BMA.